Folding, Misfolding, and Function of PMP22 Peripheral myelin protein 22 (PMP22) is a tetraspan integral membrane protein that is abundant in myelin of the peripheral nervous system (PNS). Genetic mutations that encode changes in the amino acid sequence of PMP22 are one cause of the common human peripheral neuropathy Charcot-Marie-Tooth Disease (CMTD). CMTD mutations promote misfolding, retention, and possible degradation of PMP22 early in the secretory pathway, leading to loss of function. This results in dysmyelination of PNS axons and the onset of CMTD. While clearly important, the function of PMP22 in myelin and Schwann cell physiology is not well understood. We recently quantitated the thermodynamic folding stabilities of a series of CMTD mutant forms of PMP22 and established that there is a roughly linear correlation between the thermodynamic stability of PMP22 and both cell surface trafficking efficiencies and patient nerve conduction velocities. In Aim 1 we will compare and contrast the folding kinetics and other properties of different CMTD mutant forms of PMP22 in order to examine whether the specific defects that trigger PMP22 instability and terminal misfolding in the cell are the same from mutant to mutant or whether different CMTD mutants have distinctly different defects beyond differences in stability. In Aim 2 we will determine how the instability of CMTD mutant forms of PMP22 is sensed by endoplasmic reticulum quality control. The cellular data from this aim will be integrated with the results of the biophysical experiments of Aim 1 to provide a unified biophysical and cell biological understanding of how defects in PMP22 are recognized by ER quality control. In Aim 3 we will determine how purified PMP22 promotes formation of myelin-like assemblies (MLAs) following reconstitution into lipid vesicles. This will explore the hypotheses that the role of PMP22 in MLA formation is closely related to at least one of its native functions and also that at least some CMTD mutants are likely to disrupt MLA formation in a manner that reflects how mutations promote dysmyelination in PNS tissue. While CMTD is a relatively common disorder (1:3000), to the best of our knowledge this is the only project in the world that includes the biophysical characterization of PMP22. Completion of the above aims will significantly advance our understanding of the folding, misfolding, and function of this myeli membrane protein under conditions of both health and disease. Results will provide direct insight into the molecular mechanisms underlying CMTD and may pave the way for future development of anti-CMTD therapeutics.